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Substrate RNA positioning in the archaeal H/ACA ribonucleoprotein complex

Nature Structural & Molecular Biology volume 14, pages 1189–1195 (2007)Cite this article

Abstract

The most complex RNA pseudouridylases are H/ACA ribonucleoprotein particles, which use a guide RNA for substrate capture and four proteins (Cbf5, Nop10, Gar1 and L7Ae/NHP2) for substrate modification. Here we report the three-dimensional structure of a catalytically deficient archaeal enzyme complex (including the guide RNA and three of the four essential proteins) bound to a substrate RNA. Extensive interactions of Cbf5 with one guide-substrate helix and a guide RNA stem shape the forked guide–substrate RNA complex structure and position the substrate in proximity of the Cbf5 catalytic center. Our structural and complementary fluorescence analyses also indicate that precise placement of the target uridine at the active site requires a conformation of the guide–substrate RNA duplex that is brought about by the previously identified concurrent interaction of the guide RNA with L7Ae and a composite Cbf5-Nop10 surface, and further identify a residue that is critical in this process.

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Figure 1: Overview of the substrate-loaded H/ACA RNP subcomplex structure.
Figure 2: Structural features of the guide and target RNAs in the complex.
Figure 3: RNA-RNA and RNA-protein interactions in the H/ACA RNP structure.
Figure 4: Guide RNA conformation and RNA-protein contacts in the presence and absence of L7Ae.
Figure 5: Effect of L7Ae on target RNA conformation.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grant R01 GM66958-01 (H.L.) and NIH grant RO1 GM54682 (M.T. and R.T.). B. Liang is a predoctoral fellow of the American Heart Association, Florida/Puerto Rico Affiliate (0615182B). X-ray diffraction data were collected from the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions for APS beamlines are listed at http://necat.chem.cornell.edu/ and http://www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

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  1. Bo Liang and Song Xue: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Biophysics, 91 Chiefton Way, Florida State University, Tallahassee, 32306, Florida, USA

    Bo Liang, Song Xue & Hong Li

  2. Department of Chemistry and Biochemistry, 91 Chiefton Way, Florida State University, Tallahassee, 32306, Florida, USA

    Song Xue & Hong Li

  3. Departments of Biochemistry and Molecular Biology, Green Street, Athens, 30602, Georgia, USA

    Rebecca M Terns & Michael P Terns

  4. Department of Genetics, University of Georgia, Green Street, Athens, 30602, Georgia, USA

    Michael P Terns

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Contributions

B.L. designed and carried out crystallographic studies of the wild-type complex, acquired fluorescence data, and contributed to manuscript preparation; S.X. carried out crystallographic studies of the D85A mutant complex and contributed to manuscript preparation; M.P.T. and R.M.T. supplied plasmids encoding H/ACA RNP proteins and contributed to manuscript preparation; H.L. supervised the project and contributed to manuscript preparation.

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Correspondence to Hong Li.

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Liang, B., Xue, S., Terns, R. et al. Substrate RNA positioning in the archaeal H/ACA ribonucleoprotein complex. Nat Struct Mol Biol 14, 1189–1195 (2007). https://doi.org/10.1038/nsmb1336

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